One of the characteristics of soluble retinal antigen (S-Ag) is its ability to induce an intense autoimmune inflammation in the eyes of experimental animals when injected in the presence of an adjuvant. This disease, called experimental autoimmune uveitis (EAU), is critically dependent on T cells and antigen processing by appropriate antigen-presenting cells (APC). In FY 1992, we further characterized the immunogenic and pathogenic sites of human S-Ag in the Lewis rat, using overlapping fragments of S-Ag, thus determining that there are several immunogenic sites in the S-Ag molecule as well as at least three immunopathogenic sites. Of these immunopathogenic sites, only one is immunodominant~a fragment located at sequence 341-360. The immunogenic sites for the Lewis rat do not correspond to the sites identified in the human. While most of the sites recognized by the rat immune system are clustered close to the C-terminal end of the molecule, in the human most of the activity is located in the N-terminal region. We also have tested the response of the immunodominant sequence, as well as the nondominant immunopathogenic sequences in several other rat strains in an attempt to determine whether factors within the major histocompatibility complex (MHC) class II as well as outside MHC class II may play a role in the establishment of an immunogenic and an immunopathogenic response. In human subjects, we have tested new means of establishing cell lines by seeding multiple wells with S-Ag primed peripheral blood cells and maintaining the cells in culture for 14 days in medium enriched in T-cell growth factors. This approach yields a low number of cell lines which can then be further expanded by additional rounds of stimulation. In addition, this approach allows us to determine the precursor frequency of cells reactive to S-Ag in the peripheral blood of patients and controls. This precursor frequency, which is of the order of 1 in 10 million circulating cells in patients, is lower in controls. The cell lines established using this technique will now be further characterized in terms of their response to fragments of human S-Ag as well as their T-cell receptor usage.